Notes on genome size in the hybrid Ranunculus % luizetii ( Ranunculaceae ) and its parents by flow cytometry

Notes on genome size in the hybrid Ranunculus × luizetii (Ranunculaceae) and its parents by flow cytometry.Flow cytometry was used to estimate the nuclear DNA content in the natural hybrid Ranunculus × luizetii and its parents. Our results indicate that the genome size of the hybrid R. × luizetii is closer to R. pyrenaeus than to R. parnassiifolius, providing an evidence of genome downsizing.

Since the first report on flow cytometry (FCM) analysis of plant material 38 years ago (Heller, 1973), applications of FCM in plant population and evolutionary biology have expanded dramatically.Nuclear DNA content, in combination with other morphological and molecular characters, can contribute to intergeneric classification, taxa delimitation or hybrid identification (Doležel et al., 2007).In the present work, hybrid detection using FCM has been employed to study the natural hybrid Ranunculus × luizetii Rouy (R. parnassiifolius subsp.parnassiifolius L. × R. pyrenaeus L.) collected from the Pyrenees [Spain, Lérida, Espot, Coll de la Creu de l 'Eixol, 43º 10' 35.4'' N / 4º 49' 24.1'' W, 2207 m, E. Cires & J. A. Fernández Prieto, 31975 FCO] in the same area where it was described by Rouy (1893).This study might contribute to a better understanding of zones of overlap between rare and closely related species, with potential applications to the field of conservation biology.Samples for FCM measurements were prepared from fresh tissues of young leaves of R. × luizetii and its parental taxa R. pyrenaeus; regarding to R. parnassiifolius, the FCM data come from Cires et al. (2010).Nuclear DNA amounts were estimated

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with propidium iodide staining, using a Cytomics FC 500 (Beckman Coulter) with 488-nm excitation from an argon ion laser.Data analysis was carried out using Cytomics RXP Analysis (Beckman Coulter, Inc. 2006), following the protocol described by Cires et al. (2009Cires et al. ( , 2010)).As a reference, unstained chicken red blood cell (CRBC) nuclei were added to the isolation buffer to ensure the ploidy level of parental plants.The 2C DNA content (2C = holoploid genome size; Greilhuber et al., 2005) was calculated as: The monoploid genome size (1Cx; sensu Greilhuber et al., 2005) of all plants was also calculated in mass values (pg) and Mbp, and at least 5000 nuclei were analysed per sample.
FCM analyses of Ranunculus × luizetii (Fig. 1) and its parental species resulted in high resolution histograms with mean coefficient of variation of G0/G1 peaks (interphase nuclei of cycling cells), ranging from 2.50 to 3.60%.Nuclear DNA contents are shown in Table 1.The 2C nuclear DNA content of 27 specimens of R. × luizetii and its parental taxa were determined using FCM, providing his-tograms with well-defined peaks of both sample and internal reference standards (CRBC) (Table 1).Statistical analyses revealed significant differences (P < 0.05) between the 2C DNA content of R. × luizetii and one of its parents, R. parnassiifolius.However, significant differences were not found if we take into account the other parental species, R. pyrenaeus (see Table 1).
In recent years, molecular approaches have suggested several mechanisms (e.g.elimination of non-coding and coding DNA sequences, homologous and illegitimate recombination, etc.) to explain genome downsizing in hybrids and/or polyploids (e.g.Brassica (Song et al., 1995); Nicotiana (Petit et al., 2007); Aegilops and Triticum (Eilam et al., 2008), as these mechanisms have been shown to operate in both diploids and polyploids (Leitch & Bennett, 2004).The interspecific hybridization event in Ranunculus × luizetii has been supported by morphological and molecular data (see Rouy, 1893;Cires et al., unpublished data).Indeed, Amplified Fragment Length Polymorphism (AFLP) and Internal Transcribed Spacers (ITS) of ribosomal DNA revealed genetic intermediateness between the hybrid samples (R. × luizetii) and the parental taxa; additionally a decrease in pollen fertility from the hybrid was observed (Cires et al., unpublished data).Although an intermediate genome size between the parents should be expected, FCM results indicate that the genome size of the hybrid R. × luizetii is closer to that of R. pyrenaeus than to that of R. parnassiifolius with an evidence of genome downsizing, and suggest that loss of DNA amount following hybrid formation may be a phenomenon more common than expected.Differences between populations were analyzed using a one-way ANOVA procedure and Dunn's method for pair-wise comparison.Means followed by the same letter are not statistically different (P < 0.05).

Figure 1 .
Figure1.One of the flow cytometric histograms obtained for Ranunculus × luizetii.The peaks marked with 1 and 2 indicate nuclei at the G0/G1 phase of the internal standard and the G0/G1 phase of the sample, respectively.The mean channel number (PI fluorescence), DNA index (DI = mean channel number of sample/mean channel number of reference standard) and coefficient of variation value (CV, %) of each peak are also given.Internal reference standard: CRBC: chicken red blood cells, 2C = 3.14 pg of DNA(Cires et al., 2009).

Table 1 .
Nuclear DNA contents of Ranunculus × luizetii and its parental species using FCM.The values are given as means with standard deviation of the mean (SD) of the nuclear DNA content (pg/2C).The 2C range of values (min.-minimum,max.-maximum) obtained for each population, the monoploid nuclear DNA content (1Cx) in mass values (pg) and Mbp, the mean coefficient of variation (CV, %), and the number of individuals measured (n) are also given.